Mammalian chromosomal replication origins contain unusual densely methylated DNA sequence islands in which all cytosine residues, including those in CpC, CpA, CpG, and CpT dinucleotides, are methylated as 5-methylcytosine [Tasheva, E.S. and Roufa, D.J., Mol. Cell Biol., in press (1994)]. Because three islands are fully methylated only in active growing cells, densely methylated islands (DMIs) appear either to regulate or be regulated by chromosomal replication origin activity. A plan to characterize the relationship between DMIs and active mammalian chromosomal replication origins is proposed. Three specific aims are detailed: 1) To investigate the cell cycle regulation of DMI methylation within the replication origin located at the 3' end of the Chinese hamster ribosomal protein S14 gene on chromosome 2q (ori S14). These studies will involve cells synchronized to specific stages of the mitotic cycle as well as G1S and G2 phase cells resolved by FACS. 2) To use in vitro mutagenesis to modify the DMI in ori S14 by deletion and point mutations and then to assess the mutations effect on the origins replication program. The latter will be accomplished using a novel tissue culture episomal DNA replication assay and/or homologous recombinational at the hemizygous RPS14 locus on CHO cell chromosome 2q. and 3) To identify, clone, and characterize nuclear proteins that differentially bind to bilaterally versus hemi-methylated and demethylated DMIs and thereby are likely to mediate interactions between the DMI and the cell's replication machinery.